This application is a request to continue studies of mechanisms for generation of partially reduced oxygen species (ROS) by pulmonary endothelium during lung ischemia. Our primary experimental model is the isolated, perfused rat lung that is continuously ventilated in order to maintain tissue ATP during global ischemia + reperfusion. Additional studies utilize cultured endothelial cell preparations. During our present period of great support, we have demonstrated generation of ROS and oxidative injury to lung that occurs during the ischemic period and have provided evidence for a new mechanism for initiation of ROS production, i.e., ROS production in association with endothelial cell membrane depolarization. ROS generation also was demonstrated in cultured bovine pulmonary artery endothelial cells and in the perfused lung in the presence of membrane depolarizing solutions (high K+) and with anoxia/reoxygenation produced by ventilating lungs with N2 and then 02. Studies with metabolic inhibitors indicated that ROS production with lung ischemia and with anoxia/reoxygenation occurred through different pathways and indicated a role for NADPH oxidase in lung ischemic injury. The specific aims of the present proposal are to: 1) evaluate endothelial generation of ROS through the use of fluorescence imaging in situ and to determine the temporal sequence between endothelial depolarization, changes in intracellular Ca++, Fe2+/3+ and ROS generation; 2) develop an artificial capillary system for the in vitro evaluation of the role of shear stress and shear stress adaptation on endothelial ROS generation with ischemia; 3) evaluate the role of the NADPH oxidase pathway as the source of ROS in endothelium using inhibitors, a knock-out mouse model, and antisense technology; and 4) evaluate augmentation of endothelial antioxidant defenses and protection against ischemia-mediated oxidative injury to the lung through the use of internalizable and non-internalizable site-directed antibodies coupled to antioxidant enzymes. This project will provide new information concerning a mechanism for initiation of lung oxidative injury, will document the presence of and a pathophysiologic role for NADPH oxidase in pulmonary endothelium, and will develop new methodology for possible prevention or treatment of oxidative lung injury.